Processing tissue to produce a biopolymer scaffold for tissue engineering

ABSTRACT

A method of forming and preserving a bioremodelable, biopolymer scaffold material by subjecting animal tissue, particularly fetal or neo-natal tissue, to chemical and mechanical processing. The process includes, but is not limited to, harvesting the tissue, optionally extracting growth and differentiation factors from the tissue, inactivating infective agents of the tissue, mechanically expressing undesirable components from the tissue, delipidizing the tissue, washing the tissue, optionally drying the tissue, optionally cross-linking the tissue not necessarily in the order described. The resulting product, EBM, is characterized by its microbial, fungal, viral and prion inactivated state. EBM is strong, bioremodelable, drapable and does not undergo calcification. EBM supplants previous inventions because of its unique method of preparation and broad applicability in tissue reengineering.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims the benefit ofpriority under 35 USC §120, of U.S. application Ser. No. 09/871,518,filed May 31, 2001, now U.S. Pat. No. 6,696,074 which is related to U.S.application Ser. No. 60/251,125, filed Dec. 4, 2000. The disclosure ofthe prior applications are considered part of (and are incorporated byreference in) the disclosure of this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of tissue engineering, and inparticular to a method of processing animal tissue including,particularly, fetal or neo-natal tissue to produce a biopolymer scaffoldmaterial named EB Matrix (“EBM”). EBM has broad applications for tissuerepair and regeneration. It can serve as a remodelable scaffold forrepair or replacement of human tissues and organs. It can be enrichedwith signaling molecules and cells before implantation, or with orwithout signaling molecules, it can attract host vessels and vascularcells as well as host parenchymal cells and immune systems cells topopulate it after implantation. Also, it can serve as a delivery devicefor signaling factors, cells or drugs.

2. Description of the Related Art

Rebuilding the human body is a significant industry. Human tissue banksand synthetic polymers do not meet the need for repair or replacement ofbody parts. High on the list of alternative sources of material used tomeet this need are animal tissues prepared in new ways that reduce theirimmunogenicity and maximize their usefulness and efficacy.

In the field of tissue engineering, the following three components areused alone or in combination to repair or create new tissue and organsubstitutes. 1) scaffolds made of naturally-occurring polymers (e.g.collagens), man-made polymers, (e.g. PTFE, Dacron, PET or polyethylene)or self-degrading, man-made polymers (e.g. PLA or PGA); 2) signalingmolecules that give developmental instructions to cells; and 3) cellshaving specific or multiple tissue building potential, often referred toas “stem cells”. Here we describe biopolymer matrices, produced by novelmethods, from animal tissues including fetal and neo-natal tissues to beused as tissue engineering scaffolds.

Man-made implant materials such as synthetic polymers, plastics, andsurface-coated metals may have different degrees of immunogenicity andsuffer from significant limitations that prohibit their broadapplications. A major limitation is that cells cannot remodel them afterimplantation. They are highly susceptible to microbial infection, andsome undergo calcification. Synthetic vascular conduits have a highincidence of occlusion after peripheral vascular bypass procedures.

There is a long history of the use of biopolymer matrices made fromprocessed human or animal tissues. Several methods of preservingcollagen-based matrices from animal tissues have been developed (U.S.Pat. No. 4,801,299, U.S. Pat. No. 5,336,616, U.S. Pat. No. 5,756,678,U.S. Pat. No. 5,916,265 and U.S. Pat. No. 5,997,895). All the methodsinclude a chemical step that either kills or eliminates cells. Sincetissues from post-natal animals or humans are the principal materialsprocessed, a fixation step using glutaraldehyde or a similar agent maybe used to mask antigenic determinants, eliminate the microbial burdenand increase strength. However, aldehydic processing effectivelydestroys any biological activity, such as cell binding sites, associatedwith the original tissue and greatly reduces or eliminates the abilityof cells to attach to it. It also eliminates binding sites forcell-synthesized products which attach to cells or to intermediates ableto bind to cells and cell products that make up the extracellular matrixby cells.

Collagen-based devices that are animal-derived and fixed withglutaraldehyde or a similar agent can not be remodeled since they arehighly resistant to metalloproteinase enzymes. Glutaraldehyde-treateddevices are known to undergo gradual calcification. Heart valves madefrom fixed animal tissues can require replacement in 5-7 years or soonerdue to calcification. The methods suggested in U.S. Pat. No. 4,801,299and U.S. Pat. No. 5,916,265 include the use of glutaraldehyde or asimilar agent for the fixation of tissue derived from a post-natalanimal source. The resulting products can not be faithfully remodeled.

While detergents or sodium hydroxide may be used to process post-natalanimal tissue (U.S. Pat. No. 4,801,299, U.S. Pat. No. 5,336,616, U.S.Pat. No. 5,756,678, U.S. Pat. No. 5,916,265, U.S. Pat. No. 5,997,895),they have not been used to process fetal or neo-natal animal tissue. Forexample, U.S. Pat. No. 5,997,895, filed on Apr. 30, 1998, provides acertified collagen dural substitute derived from post-natal animaltissue that undergoes an alkaline/salt treatment involving sodiumhydroxide and sodium sulfate (preferably in an aqueous solution of 5%sodium hydroxide and 20% sodium sulfate). A method for processingcollagen containing materials which uses 1.0 N sodium hydroxide wasdisclosed in a journal article in 1989 by Diringer H. and Braig H. R.(Diringer H. and Braig H. R., 1989, Infectivity of unconventionalviruses in dura mater. The Lancet, 439-440). This reference was cited inthe FDA's Guide for 510(k) Review of Process Dura Mater (1990, 2).

The product of this invention, EBM, is unlike the other products citedabove which in general are not bioremodelable. EBM is processed in a waythat preserves its binding sites for cells and cell-secreted productsthat make up the extracellular matrix surrounding cells that occupy thescaffold. EBM is also distinguished by the fact that undesirable tissuecomponents, such as DNA, are expressed mechanically from the tissue andthat delipidyzing organic solvents are used to reduce the presence ofcell and nuclear membranes. EBM does not calcify, making it safe for usein the human body for repair of soft tissues. In addition to its use forsoft tissues, EBM can be used as a scaffold for bone repair if treatedwith an appropriate growth factor, if seeded with bone precursor cellsor if occupied by bone forming cells when implanted.

EBM can be used as a tissue-building component with or without cells orsignaling complexes for creating human body replacements. It can be usedafter the addition of signaling molecules, which will further promotetissue repair. It can also be implanted after stem or differentiatedcells are seeded into or onto it.

BRIEF SUMMARY OF THE INVENTION

By processing animal tissue, including fetal or neo-natal tissue, by themethod embodying the invention, tissue strength is preserved withoutreducing its intrinsic biological properties or compromising the abilityof cells that occupy the tissue to remodel it. In addition to chemicalprocessing, a step of mechanically expressing undesirable tissuecomponents from the tissue is a significant innovation. Additionally,the uniqueness of this invention is that it includes the use of fetal orneo-natal tissue that, depending on age, is much less antigenic thanadult tissue. The present invention overcomes the difficulties inherentin the approach to animal tissue use based on glutaraldehyde treatment.

DETAILED DESCRIPTION OF THE INVENTION

For convenience, certain terms used in the specification, examples, andappended claims are collected here alphabetically.

The term “bioremodelable” or “bioremodelability” refers to a materialthat lends itself to the breakdown by cells that occupy it and use it asa template for creating a replacement made up mainly of newlysynthesized components secreted by the cells.

The terms “delipidizing” or “delipidized” refers to the removal oflipids from the tissue.

The term “DHT” refers to a dehydrothermal process, wherein the tissue iscross-linked and dehydrated at a high temperature.

The term “drapability” includes the capacity of the material to mold toirregular, curved surfaces or surfaces of other geometries.

The terms “inactivating” or “inactivated” refers to the reduction of theconcentrations of infective agents (e.g. bacteria, molds, viruses andprions) by 4, 6 or 8 logs consistent with the requirements needed toinsure against infectivity.

The phrase “mechanically expressing” refers to mechanically applyingpressure to express undesirable components from the tissue. With the aidof appropriate solvents, unwanted components from the product that arepotentially antigenic, such as DNA, RNA or other molecules released byreagents, such as NaOH, are removed.

The term “suturable” includes the ability to suture the material,wherein the material offers the required resistance to suture pull-out.

This invention involves preserving a naturally occurring,biopolymer-based matrix (EBM) from animal tissue, particularly fetal orneo-natal tissue. It provides a method of producing EBM, including thefollowing steps: (1) removing the tissue from its source; (2) optionallyextracting growth and differentiation factors from the tissue; (3)inactivating infective agents of the tissue; (4) mechanically expressingundesirable components from the tissue; (5) washing the tissue forremoval of chemical residues; (6) optionallydrying; and (7) optionallycross-linking the tissue after chemical and mechanical treatment.

In the preferred embodiment, porcine or bovine tissues, including fetaland neo-natal tissues, are used. Preferably, for example the fetalbovine tissue source is between 10 weeks of age and newborn age. As anexample, fetal bovine skin is flash frozen and stored. Other sourcematerial include blood vessels, other tubular structures, internalorgans including the bladder, tendons, ligaments, cartilage, membranessuch as the kidney capsule or diaphragm, or hard tissues such ascartilage or bone. After thawing, the tissue or organ is kept cold andchilled in an ice bath at a temperature between −4° C. and

In a preferred embodiment, a salted ice bath is used to chill the tissueto a temperature below 0° C. The tissue adhering to the underside of theskin for example is mechanically removed.

Whereas, certain desirable naturally-occurring components of tissues,particularly fetal and neo-natal tissues, may be lost because of theharsh chemicals used for viral and prion inactivation and removal ofunwanted structures and chemical components, some at high temperatures,desirable components such as growth and differentiation factors may beextracted from the skin or other tissue before the bleach and sodiumhyrdroxide treatments for the purpose of viral and prion inactivationand for removal of unwanted structural and chemical components. In analternative embodiment, growth and differentiation factors are extractedfrom the tissue by methods disclosed in U.S. Application No. 60/251,125,filed on Dec. 4, 2000, herein incorporated by reference (e.g. buffer,enzyme or acid extraction). The extracted growth and differentiationfactors, being in solution, are treated much more mildly with the agentsused for viral and prion inactivation, such as with 1 N sodium hydroxidefor 4 hrs. on a shaker on ice. After treatment, the extracted growth anddifferentiation factors are returned to the skin or other tissue beingprocessed which readily absorbs them.

In the preferred embodiment, the skin undergoes microbial, fungal, viraland prion inactivation, beginning with a treatment with bleach. Thebleach is at a concentration of between 0.05% and 5%, and the time oftreatment can vary between 1 minute and 5 hours. This step can also bedone after the sodium hydroxide step described below. All solutions arechilled with ice or salted ice to a temperature between −4° C. and 10°C.

In the preferred embodiment, the tissue is washed extensively with wateror physiological buffers (e.g. Tris-, HEPES, PBS buffer) to remove anyresidual bleach. The tissue is treated further with sodium hydroxide orpotassium hydroxide at a concentration of between 0.1 N and 10 N forbetween 10 minutes and 2 hours. This treatment also inactivatesinfective agents of the tissue (e.g. bacteria, molds, viruses andprions). The container and all solutions are chilled with ice or saltedice to a temperature between −4° C. and 10° C. The container issubsequently placed on a shaker.

In the preferred embodiment, unwanted components from the tissue (e.g.DNA, RNA or other molecules released by reagents such as NaOH) aremechanically expressed by means of repeated applications of pressureusing a flat blade (like a putty knife) and/or roller(s). The steps ofmechanically expressing material dissociated from the tissue chemicallycan be carried out by a machine as well, through an operation similar tothat used manually.

Any organic solvent as well as mixtures of them suitable for dissolvinglipids may be used for removal of lipid materials (e.g. chloroform,acetone, ether, alcohols and their mixtures). In the preferredembodiment, the tissue is delipidized in a chloroform and ethanolmixture (1:1 concentration ratio) for between 5 minutes and 5 hoursfollowed by washes in 70% ethanol and water, or the foregoing solventscan be used seriatim with the ethanol being at a concentration of 70%for similar periods of time applied after the chloroform step.

In the preferred embodiment, the tissue is subjected to extensivewashing with distilled water or buffers until the chemical residue isremoved. The final product that is designated as EBM, is stored indistilled water or buffers, or dried.

In an alternative embodiment, EBM can be cross-linked with genipin orDHT. If DHT is used, the tissue undergoes dehydration at a hightemperature.

In an alternative embodiment, EBM can be freeze-dried by rapidlyfreezing the tissue and then dehydrating it in a high vacuum.Freeze-drying increases the porosity and flexibility of the tissue.

EXAMPLE 1 Preparation of EBM

To prepare the preferred embodiment of EBM, the following protocol isfollowed. All of the following steps are performed in a laminar-flowhood using aseptic techniques and sterile solutions.

A piece of fetal bovine skin is cut out and a mark is made todistinguish the sides. The piece of skin is approximately 25×20 cm insize and is free of pigment or holes. The skin is dipped in a beakercontaining 2.0 liters of Mili-Q™ water to rinse off excess blood. Theskin is placed epidermal side down on a flat, plastic plate. The skin isflattened onto the plate.

Flesh is removed from the under side (dermal side) of the skin usingdissection tools, if done by hand; serrated tipped forceps are used tolift the flesh, and curved scissors are used to remove approximately5-10 mm wide continuous strips of flesh from one end of the skin to theother end. Defleshing, then removal of epidermis from the outer side ofthe skin can also be done by a defleshing machine. If carried outmanually, the process should continue as follows.

The skin is placed in 20% bleach for 30±3 minutes on ice. A 1.0 litersquare, wide-mouth bottle with a screw cap containing 500±50 mls ofbleach provides approximately 1.0 ml of solvent per cm² of skin. Thetemperature of the bleach is 4±2° C. The bottle is placed on a rockingplatform. The skin is then washed in 2.0 liters of Mili-Q™ water for 20minutes to dilute out the bleach.

The skin is placed on a flat, plastic plate epidermal side down. Anyremaining subdermal flesh from the bottom side is removed with a flatblade like a putty knife by applying pressure to the blade as it isdrawn over the surface of the tissue. This step mechanically expressesundesirable, chemically separated components from the tissue. The skinis held in place by a slip-resistant surface. The skin is turnedepidermal side up and the epidermis is removed with a flat blade instrips of about 5.0 mm. A machine, through an operation similar to thatused manually, can also perform the step of mechanically expression.

The skin is placed in a 5.2N solution of NaOH on a shaker on ice for15±3 minutes. A 1.0 liter square, wide-mouth bottle with a screw capcontaining 500±50 mls of NaOH provides approximately 1.0 ml of solventper cm² of skin. The temperature of the NaOH is 4±2° C.

The NaOH step is repeated twice with mechanical expression carried outbetween the steps and after the second step. The concentration of NaOHand the time of exposure to the NaOH can be increased or decreaseddepending upon the thickness of the skin. This is followed by threewashes in 1.0 liter of Mili-Q™ water for up to 20 minutes each andadditional steps of mechanical expression as needed.

As stated before, any organic solvent, as well as mixtures from them,can be used for removal of lipid material. In the preferred embodiment,a 1:1 chloroform ethanol solution treatment is followed by a 70% ethanolwash. This is followed by two washes in 1.0 liter of Mili-Q™ water forup to 10 minutes each with additional steps of mechanical expression asneeded.

EBM is immersed in 1.0 liter of sterile PBS (phosphate buffered saline)for 20+4 hours on a rocking platform at a temperature of 4±2° C. whichprovides approximately 2.0 ml of PBS per cm² of skin.

EBM may be air-dried between two porous plates with or without theapplication of pressure for 24 hours. If increased porosity is desired,EBM can be freeze-dried.

EXAMPLE 2 5 Use of EBM

EBM made from skin tissues can be used as a skin wound dressing or askin replacement tissue. With or without the addition of signalingmolecules and cells, EBM promotes wound healing. EBM is suitable for thetreatment of chronic topical wounds such as burns, ulcers, and avulsioninjuries. In grafts to host animals, such as rats, to replace fullthickness skin wounds, acellular EBM is shown to remodel to replacementskin without scaring. However, secondary derivatives are absent. Ifseeded with dermal fibroblasts and keratinocytes, EMB can serve as aliving skin replacement.

EBM can be used as a repair or replacement device throughout the humanbody.

For example, EBM can be used as a urethral sling because of its highphysical strength, resistance to stretch, suturability,cell-compatibility and bioremodelability.

EBM produced from fetal or neo-natal animal skins (e.g. porcine skin,bovine skin) can also be used for pericardial, periosteal, rotator cuff,or dura repair or replacement, or for hernia repair. It is drapable andhas single suture-pullout strength of more than 20 Newtons.

EBM can be homogenized for use in an injectable form or as a foam inhemostats, dura replacement, and other similar areas of treatment. Thesame or similar processing described in the example above can be appliedto non-skin tissues of the body to provide scaffolds of replacementparts with or without the addition of cells, signaling complexes ordrugs, with the expectation that if acellular or cellular they will bevascularized and populated with host cells.

This disclosure is not limited to preserving EBM derived from thosetissues or organs described herein. EBM may be derived from a widevariety of tissues and organs.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, issued patents, andpublished patent applications cited throughout this application arehereby incorporated by reference. The appropriate components, processes,and methods of those patents, applications and other documents may beselected for the present invention and embodiments thereof.

1. A method for producing a biopolymer scaffold, comprising: (a)providing tissue from an animal; (b) treating the tissue with KOH orNaOH; (c) applying pressure to the tissue to release componentsdisassociated from the tissue by treatment with KOH or NaOH; (d) rinsingthe tissue in aqueous solution; (e) treating the tissue with organicsolvents to delipidize the tissue; (f) applying pressure to the tissueto release components disassociated from the tissue by treatment withorganic solvents; and (g) rinsing the tissue in an aqueous solution. 2.The method of claim 1, wherein the tissue is bovine.
 3. The method ofclaim 1 wherein treatment with organic solvents comprises treatment witha solution comprising a solvent selected from the group consisting of:chloroform, acetone, ether, alcohols, and mixtures thereof.
 4. Themethod of claim 1 wherein treatment with organic solvents comprisestreatment with a mixture of chloroform and ethanol.
 5. The method ofclaim 1 wherein the tissue is rinsed with an aqueous alcohol solutionafter treatment with organic solvents.
 6. The method of claim 1 furthercomprising: (h) freeze drying the tissue.